**3. Results**

#### *3.1. Overall Structure of Salivary, Mucosal, and Fecal Bacterial Communities*

To investigate the shift in structure and composition of the mucosal, salivary, and fecal bacterial communities across patient groups, 209 out of 249 expected samples (77 mucosal, 76 salivary, and 56 fecal) underwent sequencing and processing, since in some cases (one ACD, two C, and three TCD) the samples displayed degradation of the nucleic acid due to poor conservation. A total of 3.5 million high-quality reads were obtained, of which 5,327,971 were for mucosal, 4,993,425 were for salivary, and 3,208,768 were for fecal samples. All the obtained sequences were classified into 5556 OTUs, representing 26 phyla, 52 classes, 89 orders, 156 familiae, 315 genera, and 186 species (spp.) (see Table S1: Taxonomic assignment across sample biotypes in the study cohort). The relative distribution of the phyla is shown in Figure 1, where a critical decrease of *Firmicutes* and *Actinobacteria* and an expansion of *Proteobacteria* at mucosal and salivary levels appear evident in all CD groups, mostly in ACD and RCD, in comparison with the C group that does not normalize in TCD. By contrast, no clear di fferences are evident at the stool level probably due to the high variability among samples. As regards the relative distribution of genera, as in Figure 2, an expansion of *Neisseria* and a reduction of *Streptococcus* in CD groups with respect to the C group emerge in both mucosal and salivary communities, whereas *Bacteroides* is the predominant one in the stool consortium.

**Figure 1.** Phylum-level classification of bacteria identified in individual mucosal, salivary, and fecal samples belonging to the five study groups: Each bar represents the relative contribution of phylum-level profiles of each subject enrolled in the study as indicated on the top of each panel (ACD: active celiac disease, C: control subjects, PCD: potential celiac disease, RCD: refractory celiac disease, and TCD: treated celiac disease). Twenty-six di fferent phyla were identified across the three biotypes and are represented by di fferent colors as indicated in the legend. The relative distribution of phyla among study groups indicates a critical decrease of *Firmicutes* and *Actinobacteria* and an expansion of *Proteobacteria* at mucosal and salivary levels in all celiac groups, mostly in ACD and RCD, in comparison with the C group that does not normalize in TCD. By contrast, no clear di fferences are evident in the stools.

**Figure 2.** Genus-level classification of bacteria identified in individual mucosal, salivary, and fecal samples belonging to the five study groups: Each bar represents the relative contribution of genus-level profiles with an abundance of at least 1% in each considered sample as indicated on the top of each panel. Sixty-nine different genera were identified across the three biotypes and are represented by different colors as indicated in the legend. An expansion of *Neisseria* and a reduction of *Streptococcus* in celiac groups with respect to controls emerge in both mucosal and salivary communities, whereas *Bacteroides* is the predominant one in the stool consortium.

The within-sample diversity (α-diversity) was evaluated by computing observed richness and the Chao1 and Shannon indices, of which the single values are shown in Table S2: α-diversity indices for each sample. Figure 3 shows a critical reduction of both observed richness and Shannon index in all CD groups in comparison to the C group at the mucosal level, while the Chao1 index yields significant differences when comparing ACD and PCD with C. The group that fails to be significantly discriminated despite the apparent reduction is the RCD one, possibly because of the small sample size. The salivary community shows the highest richness and Chao1 index in ACD and the lowest in RCD, while PCD and TCD display values similar to the C group. At variance with salivary samples at the fecal level, only the Shannon index produces significant differences between groups, with ACD and TCD showing higher values than C.

The comparison of β-diversity reveals significant differences only in mucosal bacterial community distribution, when considering all groups of patients (Figure 4A) and the following pairwise comparisons: TCD versus ACD (Figure 4B), and ACD versus C (Figure 4C).

**Figure 4.** Beta-diversity comparison for the mucosal microbiota: Multidimensional Scaling (MDS) plots of mucosal samples for which the Permutational Multivariate Analysis of Variance test detected a significant separation of study groups in terms of bacterial community composition. The microbiota phylogenetic distances were evaluated through the generalized UniFrac distance. Each point represents the microbiota composition of one sample. Panel (**A**): all patient groups were compared. Panel (**B**): TCD versus ACD comparison. Panel (**C**): ACD versus C comparison. Each point represents the microbiota composition of one sample.

#### *3.2. Taxonomy-Based Analyses of Mucosal Community*

As shown in Table 3, the phylum distribution is indicative of a decrease in *Actinobacteria* and an increase in *Proteobacteria* in all celiac groups, with ACD being that with the greatest deviation with respect to C. A different pattern is observed for *Bacteroidetes* that are decreased in ACD and increased in the other CD groups in comparison to C. Although *Firmicutes* are the most abundant phylum, accounting for a portion of bacterial diversity ranging from a maximum of 43.6% in C to a minimum of 34.8% in ACD, with the other CD groups displaying intermediate values (39.5% in PCD, 37.5% in TCD, and 40.16% in RCD), no significant differences are seen. Moving on to bacterial families within the last phylum, the *Streptococcaceae* show the lowest value in TCD, the *Veillonellaceae* are the lowest in ACD, and the *Lachnospiraceace* are the lowest in both ACD and RCD, whereas a critical decrease of *Gemellaceae* in PCD, ACD, and TCD in comparison to C and RCD is clearly evident. Within *Bacteroidetes*, only the *Prevotellaceae* show differences, with ACD displaying a decrease and the other CD groups displaying an increase in comparison to C. Concerning *Actinobacteria*, the *Micrococcaceae* show a critical reduction in CD groups, achieving the nadir in ACD, whilst RCD mirrors C. Finally, within the *Proteobacteria*, *Neisseriaceae* show a robust increase in all CD groups, mostly in ACD, in comparison with C. This situation is perfectly overlapped by the profile observed for the genus *Neisseria*. Among the other genera, *Streptococcus* spp. presents an opposite trend, since it is reduced in CD, especially in ACD and, to a lesser extent, in TCD, while the relative abundance found in PCD and RCD was closer to the profile detected in C. Within the same phylum of *Firmicutes* (*Peptoniphilaceae* family), *Parvimonas* spp. shows a particular profile, with a value in PCD double the level found in C, while it is the half of the C group in the other CD groups. At the species level, it is interesting to note that *Prevotellae* increase in all CD groups with respect to C, except in ACD. By contrast, *Rothia aerea* displays values in CD groups similar to C, except in PCD, where its relative abundance is decreased, and in RCD, where it appears increased.


**Table 3.** Taxa displaying significantly different relative abundances in mucosal biopsies.


**Table 3.** *Cont.*

Abbreviations: C = controls; PCD = potential celiac disease, ACD = active celiac disease; TCD = treated celiac disease; RCD = refractory celiac disease. \* The following comparisons were performed: C versus PCD; C versus TCD; C versus ACD; C versus TCD versus ACD; C versus ACD; PCD versus ACD; PCD versus ACD versus C; TCD versus RCD; ACD versus TCD; and ACD versus RCD.

#### *3.3. Taxonomy-Based Analyses of Salivary Community*

Also, in this ecosystem, *Firmicutes* is the most represented phylum, accounting for 33% of the bacterial diversity in C and falling to around 25% in TCD, ACD, and PCD and to 17% in RCD. *Bacteroidetes*, on the other hand, show more homogeneous values, ranging from 30.04% in RCD to 36.6% in TCD, with the other groups being around 33%. Interestingly, the *SR1* phylum displays a relative abundance <1% in both C (0.96%) and RCD (0.78%), whilst it rises to almost 5% in the other CD groups (4.68% in PCD and TCD and 4.23% in ACD). The comparisons between the relative abundances of taxa that yielded statistically significant differences are presented in Table 4. Within the phyla, *Proteobacteria* are increased in CD, especially in RCD, with respect to C. At the family level, again, the RCD condition differs from the other CD groups since it displays a marked reduction of *Fusobacteriaceae* and *Lachnospiraceace*. Noteworthily, within genera, *Neisseria* spp. presents a trend overlapping what was observed at the mucosal level, being critically increased in PCD, ACD, and RCD and close to C in TCD.


**Table 4.** Taxa displaying significantly different relative abundances in saliva samples.

#### *3.4. Taxonomy-Based Analyses of Fecal Community*

From Table 5, it emerges that TCD and RCD display values of relative abundances of phyla similar to C whereas ACD invariably shows a profile significantly different from TCD, except for *Proteobacteria*. At the family level, all CD groups are characterised by an increase of *Ruminococcaceae*, while *Veillonellaceae* appear decreased in ACD. Moreover, PCD displays an increased abundance in *Erysipelitrichaceae*, while RCD has an increase in *Pasteurellaceae*, which are suppressed in the other CD groups in comparison to C. Within genera, *Blautia*, *Coprococcus*, and *Roseburia* spp. show a strong increase in ACD in comparison to all the other groups, together with *Ruminococcus* spp. that appears increased even in PCD. Interestingly, *Veillonella* spp. and *Haemophilus* spp strongly increase in RCD while *Bifidobacterium* spp. and *Parabacteriodes* spp. abundance is negligible with respect to all the other groups. Finally, at the species level, the grea<sup>t</sup> increase of *Faecalibacterium prausnitzii* and *Veillonella dispar* emerges in RCD, whilst *Bifodobacterium longum*, that is increased in both PCD and ACD, appears suppressed. Also, *Roseburia faecis* appeares increased in ACD in comparison with other groups, at variance with *Bacteroides eggerthii* that displays negligible levels in both ACD and RCD, whilst it is increased in TCD.


**Table 5.** Taxa displaying significantly different relative abundances in stool samples.


**Table 5.** *Cont.*

In summary, the relative distribution of the most abundant phyla and genera in the study cohort is represented in Figures 5 and 6, respectively. It becomes evident that the salivary profiles mirror the condition at the mucosal level better than the fecal ones. The distribution of the five most abundant families is shown in Figure S1: Plot of relative abundances of the five most abundant families retrieved in each sample biotype, where, again, *Neisseriaceae* represents the most abundant one in both ACD and RCD mucosal and salivary samples.

**Figure 5.** Plot of relative abundances of the five most abundant phyla retrieved in each sample biotype: At mucosal level (upper panel), although *Firmicutes* is the most abundant phylum, it is reduced in the ACD group as *Actinobacteria*. A parallel increase of the *Proteobacteria* is found in this group, while an expansion of *Bacteroidetes* in the RCD group and a reduction of *Fusobacteria* in the PCD one are evident. In the salivary samples (central panel), the most abundant phylum is *Bacteroidetes*. Again, *Firmicutes* is reduced in the ACD group as *Actinobacteria* in comparison to the C group. Interestingly, the RCD group shows a decrease of all phyla but *Proteobacteria*. In the stool samples (lower panel), the two most abundant phyla are *Bacteroidetes* and *Firmicutes*, with the other phyla showing negligible values. Noteworthily, the former is decreased in the ACD group and increased in the TCD group in comparison to C, while the latter is increased in the ACD and PCD groups.

**Figure 6.** Plot of relative abundances of the five most abundant genera retrieved in each sample biotype: At the mucosal level (upper panel), all the most abundant genera are decreased in the ACD group but *Neisseria* as in all the other celiac groups with respect to the C one. Interestingly, *Prevotella* abundance is increased in the PCD, RCD, and TCD groups. In the salivary samples (central panel), again, *Neisseria* abundance is critically increased in all celiac groups with respect to the C one while *Prevotella* is increased only in the PCD, RCD, and TCD groups. Finally, in the stool samples (lower panel), the most abundant genus is *Bacteroides* that is increased in all celiac groups, with the other genera showing negligible values. Notably, the *Haemophilus* and *Veillonella* genera are critically increased only in the RCD group while the *Prevotella* is absent in the PCD group that displays a robust increase of the *Ruminococcus* as in the ACD group.
